Dynamic rearrangements of the actin cytoskeleton are critical to numerous cellular processes and are defective in many genetic and infectious diseases. This grant has supported work to understand actin regulatory pathways connecting Rho GTPases to Wiskott-Aldrich Syndrome Protein (WASP) family members to the Arp2/3 complex, a key actin nucleation factor. In the previous period we examined two assemblies, WRC and SHRC, that harbor the WASP proteins WAVE and WASH, respectively. We determined a preliminary cryoEM reconstruction of a WRC-Rac GTPase complex, which after refinement will reveal how Rac activates the WRC. We discovered a new WRC-binding peptide motif, and used multi-disciplinary analyses to show that it exists in >100 cell surface receptors and plays important developmental roles in flies and worms. Finally, we showed that SHRC is recruited to retromer-coated endosomes through a repeated motif in its Fam21 subunit, and that SHRC is activated in a novel fashion through WASH ubiquitination. Here, we move downstream from actin regulatory proteins to actin itself. Crystallographic and biochemical data suggest that actin monomers are highly dynamic, and that these dynamics are controlled by bound adenine nucleotides and functionally important. Yet tools have not been available previously to measure actin dynamics across the whole molecule or on a range of timescales. We recently developed eukaryotic expression protocols that enable, for the first time, actin to be labeled with the range of isotopes necessary for high-resolution NMR analyses of dynamics, including uniform 2H- and 1H/13C-methyl-labeling. These new reagents will enable us to answer long-standing, important questions about the structure and function of actin. We will use CEST-, relaxation dispersion- and T1?-based measurements to quantify dynamic parameters in ATP- and ADP-bound actin (populations of, and rates of transition between, ground states observable by crystallography and weakly populated excited states; excited state chemical shifts), and use these to structurally characterize the excited states. This will reveal the network of residues that allosterically communicate in actin, and how this communication is controlled by nucleotide. We will use the same approaches to examine changes in actin structure and dynamics induced by ligands including profilin, cofilin, DNAseI, WH2 motifs and the actin nucleation factors Bni1p and VopL. This will deepen our understanding of allostery in actin, explain the differences between sequestering and filament promoting WH2 motifs and reveal whether nucleation factors promote filament-like conformations in actin monomers. Finally, we will generate mutants that differentially sample the excited states of actin (quantified by NMR), and measure kinetic and thermodynamic properties of the corresponding filaments. Quantitative correlations between the dynamics and biochemical activities of actin will establish the functional importance of the dynamic properties. These investigations will provide a unique and deep understanding of actin that has not been available through previous crystallographic and other physical analyses.